The cornea expresses in abundance and also in a taxon-specific pattern, several cytosolic enzyme/proteins that have been named 'corneal crystallins', based on the similarity with the lens crystallins. Aldehyde dehydrogenase 3A1 (ALDH3A1) was the first abundantly expressed corneal protein identified and since then several proteins including ALDH1A1 and transketolase. We and others have provided evidence that expression of corneal crsytallins is markedly upregulated during development and differentiation as cells exit the cell cycle. Furthermore, corneal injury leading to upregulation of cell proliferation is associated with loss in expression of corneal crystallins and increased cellular light scattering and corneal haze. More recently we have found that ALDH3A1 over-expression causes both a profound retardation in cell proliferation. Our working hypothesis predicts that corneal crystallin proteins (ALDH3A1/1A1) protect against stress-induced oxidative damage by maintaining cell quiescence through growth arrest of differentiated corneal cells. In the present proposal we aim to delineate a novel role of corneal crystallins in modulating corneal cell growth as a contributing factor to corneal transparency through the following Specific Aims: Specific Aim 1: Test the hypothesis that corneal crystallins inhibit epithelial and keratocyte cell growth by modulating expression of cell cycle regulatory proteins. Human corneal epithelial and keratocyte telomerase immortalized cell lines (hTCepi and hTK) will be stably transfected with a tetracycline-inducible mammalian expression vector carrying cDNAs of wild type or mutated ALDH3A1/1A1, and measuring cell proliferation and the expression pattern of cell cycle regulating proteins. Specific Aim 2: Test the hypothesis that corneal crystallins inhibit cellular light scattering. Using a novel artificial stromal chamber that has been developed by us, the effect of crystallin protein expression on cellular light scattering and response to oxidative stress by UV-light and H2O2 will be determined. Specific Aim 3: To test the hypothesis that corneal crystallin play an important role in maintenance of corneal transparency using transgenic knockout animals. Using single and double Aldh1a1 and Aldh3a1 knockout mice the effects of crystallin protein expression on cell proliferation and transparency will be studied by assessing cell-cycle dependent protein expression and measurement of corneal haze.